15 research outputs found
Untersuchungen zur Regulation der Aktivierung humaner T-Lymphozyten bei Arginindepletion
No full textDie Verstoffwechslung der semiessentiellen Aminosäure Arginin ist entscheidend beteiligt an der Regulation der Immunantwort im Verlauf von Infektionen, entzündlichen Erkrankungen und bei Tumorwachstum. Arginin wird durch die Enzyme Arginase und Stickstoffmonoxid-Synthase abgebaut, wodurch die Verfügbarkeit der Aminosäure reguliert wird. Eine lokale Arginindepletion, welche z.B. durch Freisetzung von Arginase 1 aus myeloischen Zellen verursacht wird, führt zur völligen Hemmung der Proliferation und zahlreicher Effektor-Funktionen aktivierter humaner T-Lymphozyten. Hierbei waren die intrazellulären Mechanismen, welche zur Suppression zahlreicher T-Zell-Funktionen bei Argininmangel führen, bei Beginn dieser Promotionsarbeit, weitgehend unklar. In der vorliegenden Arbeit wurde der Einfluss von Argininmangel auf die intrazelluläre Signaltransduktion in aktivierten primären humanen T-Zellen gesunder Blutspender in vitro untersucht. Durch proteomische Analysen konnte gezeigt werden, dass das Fehlen der Aminosäure Arginin bei T-Zell-Aktivierung zu einer signifikant reduzierten Dephoshorylierung des Aktin-bindenden Proteins Cofilin führt. Normalerweise führt die Stimulation über den T-Zell-Rezeptor und kostimulatorische Moleküle via Dephosphorylierung zur Aktivierung von Cofilin, welches eine entscheidende Rolle bei der Reorganisation des Aktin-Zytoskeletts, der T-Zellproliferation und Zytokinsekretion spielt. Im Zusammenhang mit der Persistenz der phosphorylierten Form von Cofilin zeigte sich bei Argininabwesenheit ein veränderter F-Aktingehalt in aktivierten T-Zellen. Außerdem korrelierte die verminderte Ausbildung der Immunologischen Synapse, der Kontaktzone zwischen T-Zelle und antigenpräsentierende Zelle, mit der fehlenden Aktin-Bindefähigkeit von Cofilin. Im Gegensatz dazu ist ein weiterer Cofilin-abhängiger Prozess, die Migration, bei Argininabwesenheit unbeeinträchtigt. Ferner konnte erstmals eine dichotome Regulation der Zytokinsynthese und -sekretion in humanen T-Lymphozyten bei Arginindefizienz gezeigt werden. Während Arginindepletion zu einer signifikanten Inhibition von Produktion und Sekretion der Zytokine IFN-g, TNF-b und IL-10 führt, zeigte sich eine unbeeinträchtigte Synthese der Zytokine IL-2, IL-6 und IL-8. Weitere Untersuchungen zur frühen (1-30 min) Signaltransduktion bei T-Zell-Aktivierung zeigten, dass der Kalziumflux unabhängig von der extrazellulären Argininkonzentration induziert wird. Auch fanden sich keine argininabhängigen Unterschiede in der frühen Cofilin-Dephosphorylierung sowie bei Aktivierung der für die Cofilin-Dephosphorylierung verantwortlichen Ras-MEK-ERK- und Ras-PI3K-Akt-Signalwege. Im weiteren Verlauf zeigte sich dann bei Argininmangel in Assoziation mit der beeinträchtigten Cofilin- Dephosphorylierung eine reduzierte ERK-Phosphorylierung bei prolongierter Akt-Phosphorylierung. Diese Alterationen der T-Zell-Signaltransduktion fanden sich gleichartig auch bei Stimulation der Zellen in einem durch humane Granulozyten-Arginase (aus humanem Eiter ex vivo gewonnen) von Arginin depletierten Milieu und waren durch Zugabe eines Arginase-Inhibitors vollständig inhibierbar. Dieses Modell unterstreicht die Relevanz der beschriebenen T-Zell-Alterationen für die Situation granulozytär dominierter Inflammation in vivo. Das durch diese Arbeit gewonnene, bessere Verständnis intrazellulärer Regulationsmechanismen humaner T-Lymphozyten bei Arginindefizienz sollte dazu beitragen, in Zukunft in die unerwünschte Immunsuppression bei Tumorerkrankungen oder chronischer Inflammation kausal pharmakologisch eingreifen zu können
Methods for Synaptic Interrogation
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
Methods for Synaptic Interrogation
No full textThis eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contac
The dendritic density field of a cortical pyramidal cell
No full textMuch is known about the computation in individual neurons in the cortical column. Also, the selective connectivity between many cortical neuron types has been studied in great detail. However, due to the complexity of this microcircuitry its functional role within the cortical column remains a mystery. Some of the wiring behavior between neurons can be interpreted directly from their particular dendritic and axonal shapes. Here, I describe the dendritic density field (DDF) as one key element that remains to be better understood. I sketch an approach to relate DDFs in general to their underlying potential connectivity schemes. As an example, I show how the characteristic shape of a cortical pyramidal cell appears as a direct consequence of connecting inputs arranged in two separate parallel layers
Arginine deficiency leads to impaired cofilin dephosphorylation in activated human T lymphocytes.
No full textThe amino acid arginine is fundamentally involved in the regulation of the immune response during infection, inflammatory diseases and tumor growth. Arginine deficiency (e.g. due to the myeloid cell enzyme arginase) inhibits proliferation and effector functions of activated T lymphocytes. Here, we studied intracellular mechanisms mediating this suppression of human T lymphocytes. Our proteomic analysis revealed an impaired dephosphorylation of the actin-binding protein cofilin upon T-cell activation in the absence of arginine. We show that this correlates with alteration of actin polymerization and impaired accumulation of CD2 and CD3 in the evolving immunological synapse in T cell-antigen presenting cells conjugates. In contrast, T-cell cytokine synthesis is differentially regulated in human T lymphocytes in the absence of arginine. While the production of certain cytokines (e.g. IFN-γ) is severely reduced, T lymphocytes produce other cytokines (e.g. IL-2) independent of extracellular arginine. MEK and PI3K activity are reciprocally regulated in association with impaired cofilin dephosphorylation. Finally, we show that impaired cofilin dephosphorylation is also detectable in human T cells activated in a granulocyte-dominated purulent micromilieu due to arginase-mediated arginine depletion. Our novel results identify cofilin as a potential regulator of human T-cell activation under conditions of inflammatory arginine deficiency
Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.
No full textTumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+) T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8(+) T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+) T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency
Author Correction: A community-based transcriptomics classification and nomenclature of neocortical cell types
No full textImpact of arginine depletion on activation of tumor antigen specific T cells analyzed by intracellular IFN -γ staining.
No full text<p>CD8<sup>+</sup> MART-1<sub>aa26–35*A27L</sub> specific T cells of 5 HD were expanded and then restimulated by coincubation with MART-1<sub>aa26–35*A27L</sub> (black bars) or irrelevant control peptide (white bars) loaded T2 cells either in the presence (+Arg, 1000 µM) or absence (−Arg) of arginine. After 24 h, cells were fixed, permeabilized and stained for intracellular IFN-γ as well as extracellular CD3, CD8, CD28, CD45RA and CCR7. After fixation cells were analyzed by flow cytometry. <b>A</b> The fractions of IFN-γ<sup>+</sup> T cells are demonstrated relatively to the corresponding activation with MART-1<sub>aa26–35*A27L</sub> peptide pulsed T2 cells in the presence of arginine, which was set as 100%. In all HD except of HD 12 the percentage of IFN-γ<sup>+</sup> T cells was significantly reduced in the absence of arginine (p<0.05). <b>B</b> An exemplary intracellular IFN-γ flow cytometry analysis (from HD 10) is demonstrated. The numbers in the quadrants show the frequency of IFN-γ<sup>+</sup> cells as a fraction of gated CD8<sup>+</sup>CD3<sup>+</sup> lymphocytes. <b>C</b> IFN-γ<sup>+</sup> CD8<sup>+</sup>CD3<sup>+</sup> T cells express the CD28<sup>+</sup> CD45RA<sup>−/low</sup> CCR7<sup>+</sup> phenotype of antigen experienced T cells. An exemplary flow cytometry analysis (from HD10) after activation with MART-1<sub>aa26–35*A27L</sub> peptide loaded T2 cells is shown.</p
Arginine-independent cytotoxicity of tumor antigen specific T cells.
No full text<p><b>A</b> Expanded MART-1<sub>aa26–35*A27L</sub> specific CD8<sup>+</sup> T cells of 3 HD were preincubated for 24 h in the presence (+Arg, 1000 µM) or absence (−Arg) of arginine and subsequently stimulated with MART-1<sub>aa26–35*A27L</sub> peptide or control (irrelevant) peptide pulsed T2 cells for 4 h in a [<sup>51</sup>Cr]-Chromium release assay. Specific lysis of peptide loaded T2 cells upon incubation with MART-1<sub>aa26–35*A27L</sub> specific CD8<sup>+</sup> T cells in various E:T ratios (1∶1–20∶1) with/without arginine was determined in duplicates. Shown is the antigen specific lysis in % which is the specific lysis investigated with MART-1<sub>aa26–35*A27L</sub> loaded T2 cells minus specific lysis with control peptide loaded T2 cells. <b>B</b> Quantitative determination of antigen specific T cells was performed by MART-1<sub>aa26–35*A27L</sub> tetramer analysis and is displayed as % of CD8<sup>+</sup>CD3<sup>+</sup> T cells (analyzed before separation into the +Arg/−Arg groups) together with the results for the [<sup>51</sup>Cr]-Chromium release assay for each individual experiment.</p
Impact of arginine depletion on cytotoxic capacity, cytokine secretion and proliferation of human CD8<sup>+</sup> CMV antigen specific T cells expanded from the natural repertoire.
No full text<p>Expanded CMV pp65<sub>aa495–503</sub> specific T cells of 2 HD (•,▴) and a CMV pp65<sub>aa495–503</sub> specific T cell clone (▪) were activated with pp65<sub>aa495–503</sub> peptide pulsed K562-A2 cells. <b>A</b> Antigen specific tumor cell cytotoxicity was analyzed by [<sup>51</sup>Cr]-Chromium release assay at various E:T ratios and peptide concentrations (100 nM, 10 nM, 1 nM) in the presence (–––) or absence (- - -) of arginine. In parallel, expanded CMV pp65<sub>aa495–503</sub> specific T cells were incubated with pp65<sub>aa495–503</sub> peptide pulsed K562-A2 target cells for 48 h (<b>B–D</b>) or 120 h (<b>E</b>) at an E:T ratio of 20∶1. Supernatant was harvested and concentrations of IFN-γ (<b>B</b>), granzyme B (<b>C</b>) and perforin (<b>D</b>) were determined by ELISA. Data in <b>A–D</b> are representative of 3 different experiments with a total of 5 independent HD. <b>E</b> Reduced proliferation of CMV pp65<sub>aa495–503</sub> specific T cells in the absence (−Arg) compared to presence (+Arg, 1000 µM) of arginine, as determined by CFSE staining, gated on all CD3<sup>+</sup> T cells within the assay mixture. One representative experiment (total: 3) is shown in (E).</p
